Multivalent epstein-barr virus-like particles and uses thereof

ABSTRACT

Disclosed are vaccine compositions comprising a VLP comprising two or more EBV envelope glycoproteins and one or more T cell antigens and methods of preventing or treating EBV infections using the vaccine compositions. Also disclosed is an expression system or a single expression vector for co-expressing two or more EBV envelope glycoproteins simultaneously to generate a VLP vaccine. The expression system may include a single vector inserted with two or more nucleic acid sequences that encode two or more EBV envelope glycoproteins linked by one or more linking sequences such that the EBV envelope glycoproteins are co-expressed simultaneously.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application No.62/559,528, filed Sep. 16, 2017, which is incorporated by referenceherein in its entirety, including drawings.

STATEMENT OF GOVERNMENT INTEREST

The present invention was made with government support under Grant No.1R21CA205106, awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND

Epstein-Barr virus (EBV) infection of over 90% of the human populationis associated with the development of several lymphoproliferativedisorders and over 200,000 cancer cases worldwide each yea^(1, 2). Theetiology of EBV across the world is varied. In low-income settings,primary EBV infection typically occurs asymptomatically during earlychildhood¹. However, in malaria-endemic regions such as equatorialAfrica, childhood acquisition poses an increased risk of EBV+ Burkittlymphoma (BL)¹. In high-income settings such as the United States ofAmerica, primary EBV infection is delayed, but it causes acuteinfectious mononucleosis (IM) in 50-70% of adolescents with EBV primaryinfection, and significantly increases the risk of developing EBV+Hodgkin lymphoma¹. EBV is also highly associated with nasopharyngeal(China) and gastric carcinomas (Eastern Asia, Eastern Europe, and SouthAmerica), reflecting the epithelial tropism of the virus¹. Amonginfected individuals, EBV remains quiescent in memory B cells¹³, but canreactivate and cause diseases under immunosuppression, as withmalaria-associated BL, post-transplant lymphoproliferative disorders(PTLDs) in EBV-naïve children and adolescents receiving EBV+ organs, andAIDS-associated B-cell lymphomas¹⁴.

Thus, EBV infection and its associated malignancies contribute to asignificant public health burden for children and adults worldwide.There is no licensed prophylactic or therapeutic vaccine against EBVinfection or its associated diseases. A panel of experts at a 2011 NIHEBV meeting concurred on the urgent need to develop an effective andsafe vaccine to both prevent and treat EBV-associated malignancies andconsequently impact public health worldwide².

Antibodies provide the first line of defense against viral infection.Neutralizing antibodies directed against EBV envelope glycoproteinsinvolved in virus entry are present in humans, can prevent neonatalinfection, and are generated in response to immunization of humans³.However, persistent EBV infection and the limited evidence of immuneselection of viral antigenic variants indicate that in vivoneutralization of EBV infection is suboptimal. Previous candidatevaccines based on viral proteins have targeted only one arm of theimmune system, either humoral using two or more glycoproteins(prophylactic vaccine) or T cell-mediated (therapeutic vaccine), andhave shown low efficacy profiles⁴. Thus, it is important to develop amultivalent EBV vaccine that elicits robust antibody and T cellresponses. The compositions and technology disclosed herein satisfy theneed in the art.

SUMMARY

In one aspect, this disclosure relates to virus-like particles (VLPs)comprising two or more EBV envelope glycoproteins and one or more T cellantigens. In some embodiments, the EBV envelope glycoproteins includegp350, gB, gp42, gH, gL, gM, gN, BMRF2, BDLF2, BDLF3, BILF1, BILF2, andBARF1. In some embodiments, the T cell antigens include EBNA1, EBNA2,EBNA3a, EBNA3b, EBNA3c, EBNA-leader protein, and LMP2. In someembodiments, the VLP further comprises Newcastle Disease virus (NDV)structural proteins including fusion (F), matrix (M), nucleocapsid (NP),or a combination thereof.

In a related aspect, this disclosure relates to a vaccine composition ora pharmaceutical composition comprising a therapeutically effectiveamount of a single VLP comprising two or more EBV envelope glycoproteinsand one or more T cell antigens. In some embodiments, the EBV envelopeglycoproteins include gp350, gB, gp42, gH, gL, and any other known EBVenvelope glycoproteins such as gM, gN, BMRF2, BDLF2, BDLF3, BILF2,BILF1, and BARF1. In some embodiments, the T cell antigens include EBVnuclear antigen 1 (EBNA1), EBNA2, EBNA3a, EBNA3b, EBNA3c, EBNA-leaderprotein, and/or late membrane protein (LMP2). In some embodiments, theVLP further comprises NDV structural proteins including fusion (F),matrix (M), nucleocapsid (NP), or a combination thereof. In someembodiments, the vaccine composition or the pharmaceutical compositionfurther comprises one or more additional pharmaceutically acceptableantigens. In some embodiments, the vaccine composition or thepharmaceutical composition further comprises one or more adjuvants. Insome embodiments, the vaccine composition or the pharmaceuticalcomposition further comprises one or more pharmaceutically acceptablecarriers.

In a related aspect, this disclosure relates to a method of preventingor treating an EBV infection or a condition associated with an EBVinfection comprising administering to a subject in need thereof atherapeutically effective amount of the VLP, the vaccine composition orthe pharmaceutical composition described above.

In a related aspect, this disclosure relates to an immunization regimencomprising administering to a subject in need thereof one or more dosesof a therapeutically effective amount of the VLP, the vaccinecomposition or the pharmaceutical composition described above.

In a related aspect, this disclosure relates to utilization of these VLPas a platform to generate dendritic cells or T cell responses in cellculture (in vitro) that can be infused as a therapeutic treatment to asubject in need thereof one or more doses of a therapeutically effectiveamount of the cell therapy.

In another aspect, this disclosure relates to an expression system forco-expressing two or more EBV envelope glycoproteins. The expressionsystem may include a single vector inserted with two or more nucleicacid sequences that encode two or more EBV envelope glycoproteins,linked by one or more linking sequences, such that the two or more EBVenvelope glycoproteins can be co-expressed simultaneously, self-cleavedand/or self-processed to assemble into certain glycoprotein complex,e.g., gH/gL complex, gp42-gH/gL complex, gB-gH/gL complex, BMRF2/BDLF2complex or synthesized mRNA. The vector can be a plasmid vector or aviral vector. In some embodiments, the linking sequences include IRESand nucleic acid sequences encoding 2A peptides that mediate ribosomalskipping and self-cleavage. In some embodiments, the vector is insertedwith a single promoter before the two or more nucleic acid sequencessuch that the single promoter controls the expression of the two or morenucleic acid sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show generation and flow cytometry analysis of stable cellsexpressing gp350-F-gB-F-gp42-WT-gL-WT-gH-F. FIG. 1A shows transienttransfection and selection of CHO cells transfected withgp350-F-gB-F-gp42-WT-gL-WT-gH-F. FIG. 1B shows flow cytometry analysisof sorted stable cells. FIG. 1C shows second flow cytometry analysis ofCHO stable cells expressing five EBV glycoproteins.

FIGS. 2A-2C illustrate the production and characterization ofEpstein-Barr virus-like particles (EB VLPs). FIG. 2A illustrates theprocess of generating stable cells producing VLPs. FIG. 2B showsrepresentative stable cells producing VLPs stained with primaryantibodies (1:200) 72A1 or E1D1 for 30 min, compared to unstained cellsand cells stained with the isotype control alone. FIG. 2C illustratesanother example of production and sucrose gradient purification ofpolyvalent EB VLPs.

FIGS. 3A and 3B show assessment of dendritic cells maturation andstimulation of CD3 T cells. FIG. 3A shows maturation of monocyte-deriveddendritic cells (DCs) at various concentration of VLPs, from 1 μg/ml to10 μg/ml. From top to bottom, the samples are unstained, 1 μg/ml VLP, 5μg/ml VLP, and 10 μg/ml VLP. FIG. 3B shows EB VLP pulsed DCs stimulationof IFN-γ production and CD8 T cells proliferation.

FIG. 4 shows characterization of gp350 EB VLPs and generation of stableCHO-gp350 cells. After the tenth passage, stable CHO-gp350-eGFP cellswere stained with 72A1 and AF594-coupled secondary antibody (red).Expression of eGFP was detected using fluorescence detector AF488(green). DAPI was used for nuclear staining. Sequential confocal imagesshowed that stable CHO cells expressed both surface gp350 andintracellular eGFP, whereas no gp350 protein was detected inuntransfected CHO cells (negative control). Visualization of 3D mergedimages confirmed the expression of gp350 and eGFP proteins on the cellsurface and intracellularly, respectively.

FIGS. 5A-5C show immunization schedule and generation of neutralizingantibodies in immunized BALB/c mice. FIG. 5A shows immunization andbleeding schedules for 6-8-week-old female BALB/c mice. A total of 9groups of BALB/c were immunized with 10 μg of purified gp350, gB-LMP2,or gH/gL-EBNA1 VLPs or combination of gp350 and gB-LMP2; gp350 andgH/gL-EBNA1; gB-LMP2 and gH/gL-EBNA1; or gp350, gH/gL-EBNA1, and gB-LMP2VLPs. Two groups were immunized with purified UV-inactivated EBV (10 μg)or TNE buffer as positive and negative controls, respectively. FIG. 5Bshows that antibody titer in sera from immunized BALB/c mice determinedusing lysate from EBV-infected AGS cells as target, and detected byELISA. For each group of the samples on D14 and D97, respectively, fromleft to right, the samples are gp350, gB-LMP2, gH/gL-EBNA1,gp350+gB-LMP2, gp350+gH/gL-EBNA1, gB-LMP2+gH/gL-EBNA1,gp350+gB-LMP2+gH/gL-EBNA1, and UV-inactivated EBV. FIG. 5C shows thatpooled terminal bleed sera from 5 animals/immunization treatment werepre-incubated with eGFP-EBV, then incubated at 37° C. for 48 h with Rajicells; EBV-EGFP+ cells were enumerated by flow cytometry. Neutralizinganti-gp350 (72A1, dotted line) served as pos. control. TNE (negativecontrol) was used to normalize percent infection. Mice immunized with acombination of all three EB VLPs (gp350, gB-LMP2, and gH/gL-EBNA1) wasthe most effective in neutralizing infection (23.8%), followed by gp350(18.7%), gB (17.9%), or UV-inactivated EBV (16.6%). From left to right,the samples are gp350, gB-LMP2, gH/gL-EBNA1, gp350+gB-LMP2,gp350+gH/gL-EBNA1, gB-LMP2+gH/gL-EBNA1, gp350+gB-LMP2+gH/gL-EBNA1, andUV inactivated EBV. Thus, only sera from mice immunized with a mixtureof all three VLPs neutralized over 50% of EBV infection (relative toneg. control) in vitro; this was more effective than gp350 (p<0.012) orall other immunogens (p<0.0001). Similar trends were observed in HEK293cells (data not shown). These results suggest that an effective EBVprophylactic vaccine requires multiple glycoproteins. (Partly adaptedfrom Perez et al. 2017¹⁷)

FIGS. 6A-6B show immunoblot analysis of transfected cells and purifiedEB VLPs. FIG. 6A shows identification of components of EBVgp350-gB-gp42-gL-gH VLPs and NDV M and full-length NP (withoutEBNA1-LMP2). FIG. 6B shows identification of components of EB VLPsgp350-gB-gp42-gL-gH and NDV M and 26 aa of NP fused to truncated EBNA1and full-length LMP2.

FIG. 7 shows the transmission electron microscopy analysis comparingmorphology of purified EB VLPs and wild type EBV (B95-8). Panels showuranyl acetate negatively stained EB VLPs which exhibit particle sizesfrom 70 nm to 100 nm in diameter, and a structure that resembles themorphology, shape and surface appearance of a purified EBV virions.

FIG. 8 shows immunization and bleeding schedule of wild type New Zealandwhite rabbits. 10-12 week-old female and male wild type New Zealandwhite rabbits (n=6/treatment) from Pocono Rabbit Farm & Laboratory, Inc.(Canadensis, Pa.) were immunized subcutaneously three times at Days 0,28, and 42 with 50 μg of purified EB VLPs(gp350-gB-gp42-gL-gH-EBNA1-LMP2) suspended in 0.2 ml TNE buffer adsorbedto 500 μg aluminum hydroxide (alum) and 50 μg monophosphoryl lipid Afrom Salmonella enterica serotype minnesota Re 595 (MPL). 50 μg ofpurified UV-inactivated EBV, or 25 μg pf purified EBV gp350 ectodomain(4-863) protein adsorbed to alum and MPL served as positive controls,while 0.2 ml TNE buffer adsorbed to alum and MPL served as a negativecontrol. To assess short-, mid-, and long-term nAb responses, rabbitswere bled seven days pre-immunization (pre-bleed), then bled at Day 14,35, 49, and 70 after primary immunization and humanely euthanized forterminal bleeding at Day 90. Total protein of EB VLPs and UV-inactivatedEBV were quantified using Micro BCA™ Protein Assay Kit (ThermoFisher)per the manufacturer's instructions.

FIGS. 9A-9D show the evaluation of EBV-specific IgG antibodies responsegenerated in EB VLPs immunized wild type New Zealand white rabbits. FIG.9A shows the coomassie stain and immunoblot analysis of recombinant EBVgp350 and gp350-specific IgG titers from diluted sera (1:100, 1:300 and1:900). FIG. 9B shows the coomassie stain and immunoblot analysis ofrecombinant EBV gB and gB-specific IgG titers from diluted sera (1:100,1:300 and 1:900). FIG. 9C shows the coomassie stain and immunoblotanalysis of recombinant EBV gp42 and gp42-specific IgG titers fromdiluted sera (1:100, 1:300 and 1:900). FIG. 9D shows the coomassie stainand immunoblot analysis of recombinant EBV gH/gL and gH/gL-specific IgGtiters from diluted sera (1:100, 1:300 and 1:900).

FIG. 10 shows in vitro neutralization assay of EBV to determineimmunized rabbits' sera neutralizing antibody responses in HEK-293cells. For each dilution group, from left to right, the samples are TNE,EB VLP, gp350, and UV-inactivated EBV.

FIGS. 11A-11C show assessment of dendritic cells maturation pulsed withEB VLPs and their ability to stimulate CD4+ and CD8+ T cells. FIG. 11Ashows maturation of dendritic cells generated from human PBMCs (EBVseropositive individuals). From top to bottom, the samples areunstained, no VLP, 1 μl VLP, 2 μl VLP, and 4 μl VLP. FIG. 11B shows CFSEproliferation of Sample No. 18. FIG. 11C shows CFSE proliferation ofSample No. 19.

FIG. 12 shows the immunization and bleeding schedules of humanizedNSG-BLT mice. 10-12 week-old humanized mice (n=5/treatment) fromUniversity of Massachusetts Medical School were immunizedintraperitoneally three times at Days 0, 28, and 42 with 50 μg ofpurified EB VLPs suspended in 0.5 ml TNE buffer adsorbed to 500 μgaluminum hydroxide (alum) and 50 μg monophosphoryl lipid A fromSalmonella enterica serotype minnesota Re 595 (MPL). Purified 50 μg ofUV-inactivated EBV, or 25 μg of purified EBV gp350 ectodomain adsorbedto alum and MPL served as positive control, while 0.5 ml TNE adsorbed toalum and MPL served as negative control. To assess short-, mid-, andlong-term nAb responses, mice were bled seven days pre-immunization,then bled at Day 14, 35, and 49 after primary immunization and thenhumanely euthanized for terminal bleeding at Day 60. Data not shown forthe evaluation of EBV-specific IgG antibodies responses generated in EBVLPs immunized huNSG-BLT mice, in vitro neutralization and the animalchallenge with EBV to assess in vivo correlates of immune protection.Total protein of EB VLPs and UV-inactivated virus were quantified usingMicro BCA™ Protein Assay Kit (ThermoFisher) per the manufacturer'sinstructions (data not shown).

FIGS. 13A-13B illustrate the cloning strategy of EBV gp350-gB-gp42-gL-gHin MVA-BAC. FIG. 13A shows PCR amplification of EBV gp350-gB-gp42-gL-gHfrom PCAGGS using primers with engineered restriction site for cloninginto transfer vector (panel 1), ligation of EBV gp350-gB-gp42-gL-gH PCRfragment into pGEM transfer vector containing a mH5 promoter, multiplecloning site (MCS) and transcription termination signal (TS) (panel 1),insertion of Kanamycin resistance marker gene (KanR) and a I-SceIrestriction site flanked by 50 bp gB duplicate sequence (panel 3), PCRamplification of mH-EBV gp350-gB-gp42-gL-gH-TS using primers with 50 bpMVA duplicate sequence overhangs (panel 4). PCR product is inserted intoMVA-BAC via a first Red recombination utilizing the 50 bp MVA overhangssequence, and the Kan selection marker is removed from the new MVAconstruct by introducing a double-strand break at the I-SceI site andfollowed by a second Red recombination between the 50 bp gB sequenceduplication (panel 5). FIG. 13B shows MVA-BAC construct (not to scale)illustrating the five gene insertion sites: (1) Del2, (2) IGR3, (3)G1L/18R, and (4-5) Del3, going in different orientations.

FIG. 14 shows a diagram of cloning and expression of EBV genes inMVA-BAC. Schematic representation of the cloning strategy used togenerate MVA-EBV constructs. EBV-gp350-gB-gp42-gL-gH VLPs and M-NP werePCR amplified from respective donor vectors using PCR with 50 bpoverhangs homologous to the 5′ and 3′ end of G1L/18R and Del3 geneinsertion sites, respectively. En Passant cloning technique was utilizedto ligate the EBV-gp350-gB-gp42-gL-gH VLPs and M-NP PCR products intothe MVA-BAC vector containing the pBelocl 1 construct and a GFP genewhich will be used as an indicator of transfection efficiency. Theresulting clone, MVA-EBV was transfected into Baby hamster kidney(BHK)-21 cell or chicken embryo fibroblast (CEF cells). Similar strategywas used to generate wild type MVA expressing EBV-gp350-gB-gp42-gL-gHand modified EBV EBNA1 and LMP2 as candidate vaccine.

DETAILED DESCRIPTION

Expression systems, vectors, vaccines for use in preventing or treatingEBV infections are provided herein. The single polyvalent EBV subunitvaccine, which is described in detail below, can stimulate both humoral(antibody) and T cell-mediated immunity, and generate both prophylacticand therapeutic antiviral responses against EBV infection andEBV-associated malignancies.

EBV uses multiple glycoproteins to initiate entry and infection of hostcells, making them potential targets for a prophylactic vaccine⁶. gp350,gB, gp42, and the gH/gL complex or BMRF2/BDLF2 complex are theattachment/fusion glycoproteins that mediate EBV entry into host cells.They are expressed on the virions and in infected cells, and stimulatehumoral and cellular immune responses in humans and in animal models.gp350 cellular receptor interactions initiate EBV attachment to B cellsand trigger endocytosis of the virions⁶. Although this interactionenhances infection, it is not essential⁷. All clinical trials to date,which used gp350 protein as the only target protein for elicitingneutralizing antibodies have failed.^(3,6-8)

Antibodies provide the first line of defense against viral infection.Neutralizing antibodies (nAbs) directed against EBV envelopeglycoproteins are present in humans, may prevent neonatal infection, andare generated in response to immunization of humans³. However,persistent EBV infection and the limited evidence of immune selection ofviral antigenic variants indicate that in vivo neutralization of EBVinfection is suboptimal. Thus, it is important to develop a multivalentEBV vaccine that triggers both arms of the immune system to elicitrobust humoral and cellular responses.

The ability of gB and gH/gL antibodies to neutralize infection is alsowell-conserved in herpes simplex virus-1, cytomegalovirus, and Kaposisarcoma-associated herpesvirus⁵. Furthermore, gB serves as fusionmachinery and gp42 and gH/gL complexes confer host cell specificity tomediate EBV entry into B cells and epithelial cells, respectively.Importantly, the gp42 protein is unique to EBV, and recombinant EBVlacking gp42 or gH does not infect either epithelial or primary Bcells^(11,12).

Even though certain functions of some viral protein subunits werestudied, selection of appropriate viral protein subunits is veryimportant and unpredictable for producing an effective vaccine. Althoughthe major EBV surface glycoprotein gp350/220 (gp350) has been proposedas an important antigen, attempts over the past four decades to developa potent gp350-based vaccine have shown limited success. In fourindependent phase I/II clinical trials, vaccination with vectorconstructs expressing gp350 or with purified recombinant non-splicingvariant gp350 soluble protein did not prevent EBV infection, althoughacute infectious mononucleosis was reduced in young adults^(3, 6-8).

Selection of an appropriate platform is also important andunpredictable. Virus-like particles (VLPs) lack the viral genome andtypically assemble from viral structural proteins, forming repetitivearrays that resemble a natural virus. As disclosed herein, this platformallows inclusion of multiple select surface glycoproteins andintracellular T-cell antigens in a polyvalent vaccine.

Similar to other herpesviruses, EBV enters various cell types usingmultiple surface glycoproteins. Thus, the inclusion of multipleglycoproteins in the vaccine is needed to overcome the limitation ofusing gp350 alone. Disclosed herein is a platform to present multipleEBV surface glycoproteins (gp350, gB, gp42, or gH/gL) to elicitantibodies which can neutralize EBV infection in vivo.

The current opinion in the field is that protection against EBV not onlyrelies on elicitation of nAbs but also induction of CD4+ and CD8+ T-cellimmune responses specific to viral latent antigens (EBNA1, EBNA2,EBNA3a, EBNA3b, EBNA3c, EBNA-LP, LMP1, or LMP2). Thus, current EBVtherapeutic vaccine candidates have focused on enhancing suchresponses⁹.

EBV nuclear associated protein 1 (EBNA1) and latent membrane protein 2(LMP2) are intracellular proteins expressed in all EBV-infected cells,including EBV-associated tumors in children and Burkitt Lymphoma tumors.EBNA1-LMP2-specific CD4+ and CD8+ T cells are frequently detected inEBV-infected individuals, and both T-cell subsets can be effective incontrolling growth of EBV-immortalized cells. Thus, disclosed herein isthe inclusion of EBNA1 and LMP2 as components of a polyvalent vaccinewhich can trigger an effective T cell-mediated therapeutic response.

The major limitations of vaccines in pre-clinical and clinical trials todate are that none of the vaccines has created sterile immunity (i.e.,complete blockage of viral infection) and that most of the strategiesonly target one arm of the immune system, humoral or T cell-mediated.Even in cases where both arms of the immune system have been targeted ina single vaccine, such as with the use of EBV DNA packagingmutants^(18, 19), the vaccine candidates have met with limitedimmunogenicity, safety concerns, and failure to induce robust CD8+T-cell responses⁴.

Thus, disclosed herein is a novel single prophylactic and therapeuticpolyvalent VLP vaccine comprising two or more EBV envelope glycoproteinsand one or more T cell antigens. In some embodiments, the VLP vaccinecomprises two, three, four, five or more EBV envelope glycoproteins. Insome embodiments, the VLP vaccine comprises two or more T cell antigens.In some embodiments, the EBV envelope glycoproteins include gp350, gB,gp42, gH, gL, and any other known EBV envelope glycoproteins such as gM,gN, BMRF2, BDLF2, BDLF3, BILF2, BILF1, and BARF1. In some embodiments,the T cell antigens include EBNA1 and LMP2 or any other EBV neoantigens.In some embodiments, the VLP vaccine can include other EBV neoantigenssuch as LMP2, EBNA3a-c etc. In some embodiments, the VLP vaccinecomprises seven selected proteins including five EBV envelopeglycoproteins: gp350, gB, gp42, gH, and gL, and two T cell antigens:EBNA1, and LMP2. In some embodiments, in addition to the EBV envelopeglycoproteins and T cell antigens, the VLP further comprises NDVstructural proteins including fusion (F), matrix (M), and nucleocapsid(NP). In some embodiments, the VLP vaccine further comprises one or moreadjuvants.

In some embodiments, disclosed herein is a single vector co-expressingtwo or more EBV envelope glycoproteins including gp350, gB, gp42, gH,and gL, with each glycoprotein separated from another glycoprotein by 2Asequence. For example, multicistronic 2A sequence is used in apCAGGS-gp350-F-gB-F-gp42-WT-gL-WT-gH-F vector. The 2A sequence can be,for example, 15 amino acids, 16 amino acids, 17 amino acids, 18 aminoacids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids,23 amino acids, 24 amino acids, or 25 amino acids in length. In someembodiments, a sequence of GAAGAGA (SEQ ID NO:1) is used to generatefusion protein between EBNA1 and LMP2 antigens, e.g., NP-EBNA1-LMP2. Insome embodiments, a full-length NP sequence or a 26-amino acid of NPsequence is used to deliver or package EBNA1 and LMP2 into the VLPs. Insome embodiments, wild-type EBNA1 is used or the EBNA1 Gly-Ala richregions are deleted before packaging.

The amino acid sequence of the full-length NP is as follows (SEQ IDNO:2):

MSSVFDEYEQLLAAQTRPNGAHGGGEKGSTLKVEVPVFTLNSDDPEDRWNFVVFCLRIAV  60SEDANKPLRQGALISLLCSHSQVMRNHVALAGKQNEATLAVLEIDGFTNSVPQFNNTSGV 120SEERAQRFMMIAGSLPRACSNGTPFITAGVEDDAPEDIIDTLERILSIQAQVWVTVAKAM 180TAYETADESETRRINKYMQQGRVQKKYILHPVCRSAIQLTIRQSLAVRIFLVSELKRGRN 240HAGGSSTYYNLVGDVDSYIRNTGLTAFFLTLKYGINTKTSALALSSLAGDIQKMKQLMRL 300YRMKGDNAPYMTLLGDSDQMSFAPAEYAQLYSFAMAMASVLDKGTGKYQFARDFMSTSFW 360RLGVEYAQAQGSSINEDMAAELKLTPAARRGLAAAAQRVSEETSSMDIPTQQAGVLTGLS 420DGGPQAPQGGSNRSQGRPDAGDGETQFLDLMRAVANSMREAPNSVQSTTQPEPPPTPGPS 480QDNDTDWGY 489

In some embodiments, the amino acid sequence of the 26 AA fragment ofthe NP is SVQSTTQPEPPPTPGPSQDNDTDWGY (SEQ ID NO:3).

In some embodiments, disclosed herein is a method of producingEpstein-Barr virus-like particles with polycistronic vector using one ormore 2A sequences in a pCAGGS-gp350-F-gB-F-gp42-WT-gL-WT-gH-F vector.The 2A sequence can be, for example, 15 amino acids, 16 amino acids, 17amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 aminoacids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acidsin length. The method can further entail generating fusion proteinbetween truncated EBNA1 and LMP2 or any other two or more EBV latentproteins using a sequence of GAAGAGA (SEQ ID NO:1) to form a fusion ofNP-EBNA1-LMP2 or NP fused to other one or more latent proteins. In someembodiments, a full-length NP sequence or a 26-amino acid of NP sequenceis used to deliver or package EBV latent proteins into the VLPs. In someembodiments, wild-type EBNA1 is used or the EBNA1 Gly-Ala rich regionsare deleted before packaging for MHC I presentation.

As demonstrated in the working examples, subunit VLPs incorporating EBVantigens were efficiently produced in Chinese hamster ovary cells, aU.S. Food and Drug Administration-approved vehicle for most biologicsproduction, and stimulate both humoral and T cell-mediated immuneresponses in vitro and in vivo. The polyvalentgp350-gB-gp42-gH/gL-EBNA1-LMP2 VLPs (EB VLPs) can generate robustprotective anti-gp350-gB-gp42-gH/gL neutralizing antibodies (nAbs) andEBNA1-LMP2-specific T-cell responses to EBV infection.

In some embodiments disclosed herein, the EBV envelope glycoproteins canbe expressed by any suitable expression vectors including plasmidvectors and viral vectors. In some embodiments, modified Ankara vacciniavector, adeno-associated viruses, or baculovirus can be used forco-expressing two or more EBV envelope glycoproteins. The individualglycoproteins can be linked by cleavage sequences such that theco-expressed glycoproteins can be self-cleaved and self-assembled intotwo or more glycoprotein complexes.

In some embodiments, the expression systems or vectors described hereininclude two or more expression cassettes, each of which includes asingle promoter and a sequence that encodes two or more EBV envelopeglycoproteins. As a result, the two or more EBV envelope glycoproteinsare co-expressed simultaneously, i.e., under control of a singlepromoter, obviating the need for multiple promoters or vectors. Incertain embodiments, each expression cassette includes two, three, four,five, or even higher numbers of EBV glycoproteins, the expression ofwhich are under control of a single promoter. In some embodiments, avector may include more than one such expression cassette.

In some embodiments, internal ribosome entry sites (IRES) can beintroduced in between nucleic acid sequences encoding two or more EBVenvelope glycoproteins that are co-expressed, flanking the sequencesencoding the two or more glycoproteins. Although IRES can be used tolink the expression of multiple genes under a single promoter, the useof multiple IRES sequences might be limited by size constraints,instability due to its relatively larger size comparing to 2A signalsequences, and/or difference in expression levels between the geneslocated before and after an IRES. In some embodiments, 2A signalsequences that encode for the 2A peptide of food-and-mouth disease virus(F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A),Thoseaasigna virus (T2A), cytoplasmic polyhedrosis virus (BmCPV 2A), orflacherie virus (BmIFV 2A) can be used to link multiple genes under asingle promoter. 2A signal sequences have been found in picornaviruses,insect viruses and type C rotaviruses. In some embodiments, aself-cleavage 2A peptide-derived sequence from Picornaviruses¹² is usedto co-express EBV envelope glycoproteins including gp350, gB, gp42, gHand gL in native form on the surface of a VLP. Bicistronic ormulticistronic expression vectors can be used to express more than onegene product within a cell. Various suitable eukaryotic cell promoterscan be used, including but not limited to, immediate-early I promoter ofhuman CMV or the chicken beta actin promoter, promoters of vacciniavirus (mH5, pSyn, P11, p7.5), etc.

Additionally, a furin cleavage site preceding the 2A signal sequencescan be incorporated to remove the 2A peptides following self-processingof the 2A-linked polyproteins. Furin is an enzyme that occurs in theGolgi apparatus and cleaves at very short signal peptides such as KKKR(SEQ ID NO:4) or RKKR (SEQ ID NO:5) motif. Furin cleavage contributes toprotein processing and maturation. These short signal peptides can beadded to the N-terminus of the 18-22 amino acid long 2A skipping signalsso that they are removed following 2A-mediated processing of the EBVenvelope glycoproteins, except for one or two remaining amino acids. Theresultant product can be even more “native.” Although it is preferredthat the 2A-linked glycoproteins are expressed all from one vectorthrough the use of one or more expression cassettes, it is also possibleto express the 2A-linked subunits from two or more separate vectors.

By exploiting the ribosomal skipping mechanism conferred by 2A peptides,an approach of co-expressing the EBV envelope glycoproteins as only oneor two self-processing polyproteins is disclosed herein. The 2Aribosomal skipping system is widely-used to express multi-proteincomplexes due to the relative small sizes of 2A peptides (18-22 aminoacids) and because it allows stoichiometric expression of the individual2A-linked subunits. In some embodiments, P2A-linked DNA sequences of twoor more EBV envelope glycoproteins are co-expressed and efficientlycleaved and transported to the cell surface. In some embodiments, theDNA sequences encoding the EBV envelope glycoproteins arecodon-optimized. In some embodiments, the co-expressed EBV envelopeglycoproteins are self-assembled into surface complexes, includinggp42-gH/gL and gB-gH/gL.

According to the embodiments described herein, an immunization regimenis provided. The immunization regimen includes VLPs comprising two ormore EBV envelope glycoproteins and one or more T cell antigens. Theimmunization regimen may be administered via prime/boost homologous(e.g. using only the same vaccine type) or heterologous (e.g. usingdifferent vaccine types) vaccination. The immunization regimen may beadministered in a dose vaccination schedule involving two or moreimmunizations, which may be administered 2 weeks to 6 months apart.Other suitable immunization schedules or regimens that are known in theart may be used according to the embodiments described herein by thoseskilled in the art.

According to some embodiments, the nucleic acid sequences encoding twoor more EBV envelop glycoproteins are assembled into a single vector,with a linking sequence inserted between the nucleic acid sequencesencoding two or more subunits. For example, the EBV envelopeglycoproteins may be linked through linking sequences such as internalribosome entry sites (IRES), derived from a number of different RNAviruses that are well known in the art and sequences encoding 2Apeptides, to link all or some of the EBV envelop glycoproteins. The 2Asignal sequence encoding a 2A peptide of foot-and-mouth disease virus(F2A), a 2A peptide of equine rhinitis A virus (E2A), a 2A peptide ofporcine teschovirus-1 (P2A), a 2A peptide of cytoplasmic polyhedrosisvirus (BmCPV 2A), a 2A peptide of flacherie virus (BmIFV 2A), or a 2Apeptide of Thosea asigna virus (T2A), can be used.

The vaccine composition as described herein may comprise atherapeutically effective amount of a VLP as described herein, and mayfurther comprise a pharmaceutically acceptable carrier according to astandard method. Examples of acceptable carriers include physiologicallyacceptable solutions, such as sterile saline and sterile bufferedsaline.

In some embodiments, the vaccine or pharmaceutical composition may beused in combination with a pharmaceutically effective amount of anadjuvant to enhance the anti-EBV effects. Any immunologic adjuvant thatmay stimulate the immune system and increase the response to a vaccine,without having any specific antigenic effect itself may be used as theadjuvant. Many immunologic adjuvants mimic evolutionarily conservedmolecules known as pathogen-associated molecular patterns (PAMPs) andare recognized by a set of immune receptors known as Toll-like Receptors(TLRs). Examples of adjuvants that may be used in accordance with theembodiments described herein include Alum, Freund's complete adjuvant,Freund's incomplete adjuvant, double stranded RNA (a TLR3 ligand), LPS,LPS analogs such as monophosphoryl lipid A (MPL) (a TLR4 ligand),flagellin (a TLR5 ligand), lipoproteins, lipopeptides, single strandedRNA, single stranded DNA, imidazoquinolin analogs (TLR7 and TLR8ligands), CpG DNA (a TLR9 ligand), Ribi's adjuvant (monophosphoryl-lipidA/trehalose dicorynoycolate), glycolipids (α-GalCer analogs),unmethylated CpG islands, oil emulsion, liposomes, virosomes, saponins(active fractions of saponin such as QS21), muramyl dipeptide, alum,aluminum hydroxide, squalene, BCG, cytokines such as GM-CSF and IL-12,chemokines such as MIP 1-α and RANTES, activating cell surface ligandssuch as CD40L, N-acetylmuramine-L-alanyl-D-isoglutamine (MDP), andthymosin α1. The amount of adjuvant used can be suitably selectedaccording to the degree of symptoms, such as softening of the skin,pain, erythema, fever, headache, and muscular pain, which might beexpressed as part of the immune response in humans or animals after theadministration of this type of vaccine.

In further embodiments, use of various other adjuvants, drugs oradditives with the vaccine of the invention, as discussed above, mayenhance the therapeutic effect achieved by the administration of thevaccine or pharmaceutical composition. The pharmaceutically acceptablecarrier may contain a trace amount of additives, such as substances thatenhance the isotonicity and chemical stability. Such additives should benon-toxic to a human or other mammalian subject in the dosage andconcentration used, and examples thereof include buffers such asphosphoric acid, citric acid, succinic acid, acetic acid, and otherorganic acids, and salts thereof; antioxidants such as ascorbic acid;low molecular weight (e.g., less than about 10 residues) polypeptides(e.g., polyarginine and tripeptide) proteins (e.g., serum albumin,gelatin, and immunoglobulin); amino acids (e.g., glycine, glutamic acid,aspartic acid, and arginine); monosaccharides, disaccharides, and othercarbohydrates (e.g., cellulose and derivatives thereof, glucose,mannose, and dextrin), chelating agents (e.g., EDTA); sugar alcohols(e.g., mannitol and sorbitol); counterions (e.g., sodium); nonionicsurfactants (e.g., polysorbate and poloxamer); antibiotics; and PEG.

The vaccine or pharmaceutical composition containing a VLP describedherein may be stored as an aqueous solution or a lyophilized product ina unit or multiple dose container such as a sealed ampoule or a vial.

The expression systems, vectors and vaccines described herein may beused to treat or prevent any EBV infection or conditions associated withEBV infection such as EBV+ lymphomas, carcinomas, PTLDs, multiplesclerosis among other diseases.

As used herein, the term “subject” is an animal. In some embodiments,the subject is a mammal. In some embodiments, the subject is human.

The term “an effective amount” as used herein refers to an amount of acomposition that produces a desired effect. For example, a population ofcells may be infected with an effective amount of a viral vector tostudy its effect in vitro (e.g., cell culture) or to produce a desiredtherapeutic effect ex vivo or in vitro. An effective amount of acomposition may be used to produce a prophylactic or therapeutic effectin a subject, such as preventing or treating a target condition,alleviating symptoms associated with the condition, or producing adesired physiological effect. In such a case, the effective amount of acomposition is a “therapeutically effective amount,” “therapeuticallyeffective concentration” or “therapeutically effective dose.” Theprecise effective amount or therapeutically effective amount is anamount of the composition that will yield the most effective results interms of efficacy of treatment in a given subject or population ofcells. This amount will vary depending upon a variety of factors,including but not limited to the characteristics of the composition(including activity, pharmacokinetics, pharmacodynamics, andbioavailability), the physiological condition of the subject (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage, and type of medication) or cells, thenature of the pharmaceutically acceptable carrier or carriers in theformulation, and the route of administration. Further an effective ortherapeutically effective amount may vary depending on whether thecomposition is administered alone or in combination with anothercomposition, drug, therapy or other therapeutic method or modality. Oneskilled in the clinical and pharmacological arts will be able todetermine an effective amount or therapeutically effective amountthrough routine experimentation, namely by monitoring a cell's orsubject's response to administration of a composition and adjusting thedosage accordingly. For additional guidance, see Remington: The Scienceand Practice of Pharmacy, 21st Edition, Univ. of Sciences inPhiladelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa.,2005, which is hereby incorporated by reference as if fully set forthherein.

“Treating” or “treatment” of a condition may refer to preventing thecondition, slowing the onset or rate of development of the condition,reducing the risk of developing the condition, preventing or delayingthe development of symptoms associated with the condition, reducing orending symptoms associated with the condition, generating a complete orpartial regression of the condition, or some combination thereof.Treatment may also mean a prophylactic or preventative treatment of acondition.

In some embodiments, the vaccine or pharmaceutical composition describedherein may be used in combination with other known pharmaceuticalproducts, such as immune response-promoting peptides and antibacterialagents (synthetic antibacterial agents). The vaccine or pharmaceuticalcomposition may further comprise other drugs and additives. Examples ofdrugs or additives that may be used in conjunction with a vaccine orpharmaceutical composition described herein include drugs that aidintracellular uptake of the composition or vaccine disclosed herein,liposome and other drugs and/or additives that facilitate transfection,(e.g., fluorocarbon emulsifiers, cochleates, tubules, golden particles,biodegradable microspheres, and cationic polymers).

In some embodiments, the vaccine composition or pharmaceuticalcomposition described herein may be administered by directly injecting aVLP suspension prepared by suspending the VLP in PBS (phosphate bufferedsaline) or saline into a local site, by nasal or respiratory inhalation,or by intravascular (i.v.) (e.g., intra-arterial, intravenous, andportal venous), subcutaneous (s.c.), intracutaneous (i.c.), intradermal(i.d.), or intraperitoneal (i.p.) administration. The vaccine orpharmaceutical composition of the present invention may be administeredmore than once. More specifically, after the initial administration, oneor more additional vaccinations may be given as a booster. One or morebooster administrations can enhance the desired effect. After theadministration of the vaccine or pharmaceutical composition, boosterimmunization with a pharmaceutical composition containing the VLP asdescribed herein may be performed.

The following examples are intended to illustrate various embodiments ofthe invention. As such, the specific embodiments discussed are not to beconstructed as limitations on the scope of the invention. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the scope ofinvention, and it is understood that such equivalent embodiments are tobe included herein. Further, all references cited in the disclosure arehereby incorporated by reference in their entirety, as if fully setforth herein.

EXAMPLES Example 1: Construction of Polycistronic Plasmid, Generationand Flow Cytometry Analysis of Stable Cells ExpressingGp350-F-gB-F-Gp42-WT-gL-WT-gH-F

Chimeric fragments of gp350 (1-864), gB (1-735), and gH (1-679) wereconstructed by fusing the ectodomain (ED) of the individual viralproteins to the Newcastle disease virus fusion protein (NDV-F)transmembrane (TM) and cytoplasmic (CT) domains. These chimericfragments, along with gp42 wild-type (1-223) and gL wild type (1-140)were encoded as a single transcript (gp350-F-gB-F-gp42-WT-gL-WT-gH-F)within a modified pCAGGS vector. Each of the viral proteins wasseparated by a short unique 2A peptide sequences, which acts as acleavage signal peptide during processing.

CHO cells were co-transfected with polycistronicpCAGGS-gp350-F-gB-F-gp42-WT-gL-WT-gH-F and pCl-puro plasmids.Forty-eight hours post-transfection, cells were cultured in mediacontaining 10 μg/ml of puromycin. Selected cells were stained withanti-gp350 monoclonal antibody (mAb) 72A1 at a concentration of 1:200,followed by staining with secondary antibody, goat anti-mouse IgGconjugated to Alexa Fluor 488 (1:1000). Stained cells were washed threetimes with phosphate buffer saline, sorted using flow cytometry andpositive cells expanded under selection of puromycin and further sortedfour times as indicated in FIG. 1A, panels i-iv. Stable CHO cellsreached approximately 95% positivity by the third sort (FIG. 1A, paneliii), and subsequently the concentration of puromycin was reduced to 5μg/ml (FIG. 1A, panel iv).

As shown in FIG. 1B, FACS analysis of cells maintained in 5 μg/ml ofpuromycin showed a decrease in the percent of positive cells whenstained with mAb-72A1, anti-gB mAb (CL55), anti-gp42 mAb (F-2-1),anti-gL mAb (E1D1), anti-gH mAb (CL59), or anti-gH/gL mAb (CL40) atconcentrations of 1:200, followed by staining with secondary antibody,goat anti-mouse IgG conjugated to Alexa Fluor 488 (1:1000) and analyzedby flow cytometry.

Stable CHO cells expressing all the five EBV glycoproteins wereenriched, stained, and analyzed as described above. During this periodanother anti-gp350 mAb 2L10 was also used. The transfected cells werecompared to unstained cells and cells stained with the isotype controlalone (data not shown). As shown in FIG. 1C, stable CHO cells maintainedin 10 μg/ml of puromycin expressed >90 percent positivity of all thefive glycoproteins incorporated. Anti-gp350 mAb (72A1, 2L10) mAbs werepurchased from Millipore; mAbs E1D1, CL55, F-2-1, CL59, CL40 were giftsof L. Hutt-Fletcher (Louisiana State University, Shreveport).

Example 2: EB VLPs in Eliciting Immune Responses in Wild Type BALB/Mice

Construction of Gp350-gB-Gp42-gH/gL-EBNA1-LMP2 VLP Plasmids:

To develop EB VLPs, pCAGGS-NDV M, and a fusion protein encoding for atruncated EBNA1 [amino acids (aa) 325-641], and full-length LMP2 fusedto 26aa of NDV NP (aa 474-489) were synthesized. Full-length NDV NP wasused previously to package EBNA1 and LMP2 inside gH/gL and gB VLPs,respectivelyl⁷. It was recently shown that aa of the NP sequence ofparamyxoviruses is sufficient to deliver foreign proteins inside aVLP²¹. Thus, the NP fragment (26aa NDV NP) was truncated tosignificantly increase the efficiency of packaging EBNA1-LMP2 into theEB VLP. Production and characterization of EB-VLPs are illustrated inFIG. 2.

To construct the gp350-gB-gp42-gH/gL glycoproteins, a chimeric fragmentencoding a polycistronic gp350-F-gB-F-gp42-gL-gH-F was synthesized, inwhich the ectodomains of g350, gB, and gH were fused to the NDV Ftransmembrane/cytoplasmic domains¹⁷. To enable expression andinteraction between gH/gL and gp42 or gB as native complexes gp42-gH/gLor gB-gH/gL, unique amino acid 2A linker sequences (18 aa) wereinterspersed between individual gene cDNAs. This provides a cleavagesite that allows gp350, gB, gp42, gL, and gH to be expressed at similarratios from a single transcript and be independently released. Thesynthesized chimera was cloned into a mammalian expression vector(pCAGGS) and verified sequence fidelity. cDNAs of full-length gp350²²,NDV M, gB, gp42, gH/gL, gp42-gH/gL, gB-gH/gL, LMP2, and EBNA1 weresynthesized and individually cloned into pCAGGS as controls forcharacterization studies.

As shown in FIG. 2A, the plasmid encoding for five EBV glycoproteinsgp350-F-2A-gB-F-2A-gp42-WT-2A-gL-WT-2A-gH-F from a single transcript wastransfected into CHO cells or human embryonic kidney 293 cells alongwith a plasmid containing a puromycin resistance gene to generate stablecells. The transfected cells were cultured in the presence of 2-10 μg/mlof puromycin. Surviving cell colonies were expanded, stained withanti-gp350 (72A1) or anti-gH/gL (E1D1) and sorted twice by flowcytometry (FC) to enrich positive stable cells. To generate VLPs, stablecells expressing >80% of all glycoproteins by FC were co-transfectedwith pCAGGS-NDV-M and full length NP plasmids or NDV-M and 26aaNP-EBNA1-LMP2 plasmids (transiently or stably, see sucrose densitygradient purified VLPs) indicating that use of 26aaNP-EBNA1-LMP2 doesnot affect efficiency of production. Representative stable cellsproducing VLPs stained with primary antibodies (1:200) 72A1 or E1D1 for30 min, followed by staining with goat anti-mouse IgG-Alexa Fluor 488(1:500) for 30 m. Cells were washed twice and analyzed by FC. Thetransfected cells were compared to unstained cells and cells stainedwith the isotype control alone as shown in FIG. 2B. Cells were alsostained with anti-gB (CL55), anti-gH or gH/gL (CL59) and anti-gp42(F-2-1) (FIG. 1).

As shown in FIG. 2C, stable CHO cells expressing glycoproteins gp350,gB, gp42-WT, gL-WT and gH from a single transcript were expanded into100 T175 flasks and cells transiently co-transfected with plasmidsencoding for pCAGGS-NDV-matrix (NDV-M) and full-length NP plasmids orpCAGGS-NDV-M and plasmid encoding for 26 amino acids of NP (463-489)fused to EBNA1 (326-641) and LMP2 (1-501) (26aaNP-EBNA1-LMP2).Supernatant from transfected cells were collected every 24 hours for 5days and EB VLPs pelleted and purified through sucrose density gradient.

Dendritic cells (DCs) were generated from monocytes isolated from humanPBMC samples. Monocytes were maintained in culture with 100 ng/ml GM-CSFand 25 ng/ml IL-4 for 7 days to induce differentiation into DCs as shownin FIG. 3A. Differentiated DCs were stained with anti-CD14 (FITC),anti-CD83 (PE), and anti-HLA-DR (APC) and analyzed by flow cytometrybefore being co-cultured with autologous purified T-cells. DCs pulsedwith EB VLPs were co-cultured with autologous T-cells labeled with CFSE(TC to DC ration of 60:1). On Day 7 T-cells were re-stimulated witheither LPS (100 ng/ml), a pool of 25 EBV-specific peptides (50 ng/ml perpeptide), or DCs freshly pulsed with EB VLPs (2.5 μl). Cultures wereanalyzed after 24 h for CD8 expression (PerCP), CFSE, and IFN-γproduction (AF-700). Brefeldin A was included in the cultures for thefinal 5 hours before analysis to block cytokines release. EB VLPsstimulated CD8 T cell proliferation were compared to unstimulated cellsor cells stimulated with LPS or EBV peptides, as shown in FIG. 3B.

Stable CHO-gp350-gB-gp42-gH/gL cells were co-transfected with equalamounts of pCAGGS-26aa NDV NP-EBNA1-LMP2 and pCAGGS-NDV M, as well aspClneo plasmid to allow selection of stable cells with both puromycinand neomycin. Upon selection with both antibiotics, five clones wereselected, amplified, and single-cell sorted (using antibodies describedabove) into 96 well-plates containing selection media (puromycin andneomycin). The sorted cells were expanded from the 96-well plate to T175flasks and supernatant from stable cells was collected between 24-96 h,and EB VLPs were purified as described²². To confirm production of gp350, gB, gp42, gH/gL, EBNA1, LMP2, and NDV components, the purified VLPscan be analyzed as described¹⁷.

Immunization of BALB/c Mice to Generate nAbs:

Five groups (n=5/treatment) of 6-8-week-old BALB/c wild-type mice or NewZealand white rabbits can be immunized intraperitoneally three times(Days 0, 29, and 54) with 12.5 μg, 25 μg or 50 μg of purified EB VLPs in0.5 ml of TNE buffer adsorbed to aluminum hydroxide (alum adjuvant usedto improve immunogenicity; 0.25 μg alum/μg protein). PurifiedUV-inactivated EBV or TNE adsorbed to alum can serve aspositive/negative controls, respectively. To assess short-, mid-, andlong-term immunogenic nAb responses, mice can be tail-vein bled toobtain serum at two-week intervals after primary immunization, untilsacrifice at Day 97.

Gp350-gB-Gp42-gH/gL-Specific Antibody Titer and Neutralization Assay:

ELISA is used to assess and compare antibody titers against EBVglycoproteins included in the vaccine in sera collected from mice, usingsoluble proteins gp350, gB, gp42, gH/gL, or gp42-gH/gL (Immune Tech) orpurified EBV lysate as binding targets. Sera can be used to conductneutralization assays against EBV-eGFP produced in either B cells(B95-8) or epithelial cells (AGS) in an in vitro system using differentcell lines and primary B cells as described²². FC is used to determinepercent of EBV-eGFP+ cells. Sera from TNE-immunized animals can serve asnegative controls.

Statistical Analysis:

Using 5 mice/dose, the minimum observed neutralizing activity serves asa simple lower 97% confidence limit on the median, so ≥70% neutralizingactivity in all 5 mice at the highest dose is observed, it is concludedthat the median neutralizing activity is significantly greater than 70%,which is regarded as a promising level of neutralization. Neutralizingactivity is reported as median and range for the five mice in eachgroup, the range serving as a 95% confidence interval. Isotonicregression (pooling non-significant order violators) is used to smoothmedians if they do not increase with dose. Antibodies titers can besummarized using a similar approach. The main comparisons of interestare between the highest dose and the UV-EBV control. Preliminary data onantibody titers gave 0.47 as the coefficient of variation (8 antigens,root mean square SD of log titers). With 5 mice/dose, this provides 80%power to detect a difference of 0.95 on the natural log scale, i.e., anantibody titer ratio of 2.6, or its reciprocal 0.38. The comparisons arepresented as the ratio of geometric means, with a 95% confidenceinterval.

Example 3: Determining the Efficacy of the EB VLP Vaccine in a HumanizedMouse Model and In Vitro

In this example, a mouse model harboring a functional human immunesystem can be used. To replicate human vaccine responses in vitro, whereVLP-pulsed human peripheral blood mononuclear cells (PBMCs) can be usedas antigen-presenting cells to expand EBV-specific CD4+ and CD8+ Tcells¹⁸.

Immunization of Humanized Mice to Generate nAbs:

huNSG-BLT mice can be used to test the ability of the EB VLP to blockEBV primary infection of human B cells and elicit EBV-specific T-cellresponses in vivo. The mice are immunized and antibody titer isdetermined as described in Example 2.

Viral Challenge in huNSG-BLT Mice:

To determine vaccine efficacy, immunized mice are challenged with ˜1×10³TD₅₀ (50% transforming dose) of EBV-eGFP intravenously through the tailvein three weeks after final immunization. The blood is collected at 0,6, 24, 48, 96, and 120 h post-inoculation. FC is used to quantify thenumber of infected B cells in vivo (EBV-eGFP+). To assess EBVreplication, at various time points, PBMCs are isolated, viral RNA/DNAis extracted, and RT-PCR and qPCR are used to detect and quantify viralDNA, using specific primers for EBV biomarkers such as EBV-encoded RNA,EBNA1, LMP1, or LMP2.

Quantification and Quality of T-Cell Responses:

To enumerate the number of EBNA1-LMP2 or any other EBV antigen-specificCD4+ and CD8+ T cells, autologous T cells co-cultured with pulsed DCsare stained using T-cell markers CD3/CD4/CD8, and EBNA1-LMP2-or anyother EBV antigen-specific pentamers (PeproTech). FC staining can beused for extracellular markers CD3/pentamer-positive populations byCD4+vs. CD8+ status, CD137+, and cytokines expressions.

Statistical Analysis:

Collecting >100,000 events can provide statistical differences byt-test. A quantitative analysis by FC can be performed to measure the invitro expansion of EB VLP-specific T cells from purified human dendriticcells pulsed with purified EB VLPs.

To assess the ability of sera from EB VLP-immunized mice to neutralizeinfection in vitro, pooled sera collected at Day 97 were used inneutralization assays in HEK-293 epithelial and Raji B-cell lines, whichare susceptible to AGS-EBV-eGFP, as evidenced by eGFP+ cells²². Virustiter and percent of eGFP+ Raji cells were determined by flow cytometry(FC) as described²², and neutralization titer was defined as 50%inhibition of infection, compared to control sera from EBV-seronegativeanimals. When 5 μl of AGS-EBV-eGFP virus was pre-incubated with seriallydiluted sera (1:1, 1:2.5, and 1:5) from TNE-immunized mice (negativecontrol), fluorescence dropped from 50% (virus alone) to 40% and wasused to normalize percent infection. In contrast, serially diluted serafrom mice immunized with EB VLPs or UV-inactivated EBV neutralizedinfection in a dose-dependent manner (FIG. 5C). Purified anti-gp350 mAb72A1 (5 μg/ml), known to block EBV infection²⁴, served as positivecontrol (dotted line). Mice immunized with a combination of all three EBVLPs (gp350, gB-LMP2, and gH/gL-EBNA1) was the most effective inneutralizing infection (23.8%), followed by gp350 (18.7%), gB (17.9%),or UV-inactivated EBV (16.6%). Thus, only sera from mice immunized witha mixture of all three VLPs neutralized over 50% of EBV infection(relative to negative control) in vitro; this was more effective thangp350 (p<0.012) or all other immunogens (p<0.0001). Similar trends wereobserved in HEK293 cells (data not shown). These results suggest that aneffective EBV prophylactic vaccine requires multiple gps.

Example 4: Immunoblot Analysis of Transfected Cells and Purified EB VLPs

Untransfected CHO cells, transfected cells, purified EB VLPs(gp350-gB-gp42-gL-gH or gp350-gB-gp42-gL-gH-EBNA1-LMP2) and purified EBVwere lysed, run on a 4-12% SDS polyacrylamide gel, and analyzed usingimmunoblot. Briefly, proteins were transferred onto immunoblot membrane,blocked with 3% BSA for one hour and followed by primary and secondaryantibody staining.

FIG. 6A shows that partial characterization of the components of EB VLPs(gp350-gB-gp42-gL-gH NDV M, and full-length NP) were analyzed bystaining the blot with anti-gp350 (72A1) left panel, or polyclonalanti-NDV middle panel and mAb anti-EBNA1 right panel. A panel to theleft (anti-gp350), untransfected CHO cells, CHO cells transfected withpCAGGS plasmids served as negative control. Purified EBV lysate andstable CHO cells expressing EBV five glycoproteins (CHO 5 in 1) servedas positive control. Middle panel, presence of NDV NP was analyzed bystaining the blot with polyclonal anti-NDV. CHO cells transfected withpCAGGS-NP (CHO-NP), -NP-EBNA1-LMP2 or -26aa-EBNA1-LMP2 served aspositive controls. Untransfected CHO cells, CHO cells transfected withpCAGGS-EBNA1 served as negative controls. In the right panel, blots werestained with anti-EBNA1 which detected various isoforms of EBNA1 in CHOcells transfected with pCAGGS-EBNA1, -NP-EBNA1, -NP-EBNA1-LMP2, andNP26AA-EBNA1 LMP2. Untransfected cells and CHO cells transfected withpCAGGS alone served as negative controls.

FIG. 6B shows complete characterization of EB VLPs(gp350-gB-gp42-gL-gH-EBNA1-LMP2). Rabbit polyclonal anti-2A, mAbanti-gp350 (72A1), or mAb anti-gH/gL (made in our laboratory) were usedto detect all EBV glycoproteins incorporated on VLPs, first three panelsto the left. Untransfected CHO cells, CHO cells transfected with pCAGGSalone, pCAGGS gp350, pCAGGS gH/gL or purified EBV served as positive andnegative controls. Anti-EBNA1 or anti-LMP2 (1467) were used to detectEBNA1 and LMP2 incorporated in the EB VLPs in the two panels to theright. Un-transfected CHO cells and CHO cells transfected withpCAGGS-NP, served as negative controls. CHO cells transfected withpCAGGS-NP-EBNA1, or -NP-EBNA-LMP2 served as positive controls. Rabbitpolyclonal anti-NDV to detect NP protein was a gift of Dr. T. Morrison,University of Massachusetts Medical School, Worcester, Mass.), andanti-DNA binding domain EBNA1 mAb was a gift of F. Grasser, Institut fürVirologie, Germany). Anti-LMP2 (clone 1467) was purchased from SantaCruz.

Example 5: Immune Responses of EB VLPs

As shown in FIG. 8, 10-12 week-old female and male wild type New Zealandwhite rabbits (n=6/treatment) from Pocono Rabbit Farm & Laboratory, Inc.(Canadensis, Pa.) were immunized subcutaneously three times at Days 0,28, and 42 with 50 μg of purified EB VLPs(gp350-gB-gp42-gL-gH-EBNA1-LMP2) suspended in 0.2 ml TNE buffer adsorbedto 500 μg aluminum hydroxide (alum) and 50 μg monophosphoryl lipid Afrom Salmonella enterica serotype minnesota Re 595 (MPL). 50 μg ofpurified UV-inactivated EBV, or 25 μg pf purified EBV gp350 ectodomain(4-863) protein adsorbed to alum and MPL served as positive controls,while 0.2 ml TNE buffer adsorbed to alum and MPL served as a negativecontrol. To assess short-, mid-, and long-term nAb responses, rabbitswere bled seven days pre-immunization (pre-bleed), then bled at Day 14,35, 49, and 70 after primary immunization and humanely euthanized forterminal bleeding at Day 90. Total protein of EB VLPs and UV-inactivatedvirus were quantified using Micro BCA™ Protein Assay Kit (ThermoFisher)per the manufacturer's instructions.

IgG titers were measured by ELISA using purified soluble gp350, gH/gL,gp42, or gB as target antigens (see commassie blue blots for purity ofthe proteins). First, 96-well microtiter plates (Nunc-Immuno PlateMaxisorp) were coated with 50 ng/well of the target antigen in PBSbuffer (pH 6.2) at 4° C. overnight and blocked with 1% BSA. Sera fromimmunized rabbits were serially diluted in PBS (1:100, 1:300, 1:900,1:2700, 1:8100), added to the plate and incubated for 2 hours at roomtemperature (RT) and the plates were washed three times. Antibodybinding was detected with HRP-labeled anti-rabbit IgG secondary antibodyafter incubation at RT for 1 hour. Plates were washed 3 times and thesubstrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; ABTSSera Care) was added. The reactions were stopped with ABTS stop solution(Sera Care). To determine antibody titer, optical density (OD) for eachprotein was read at 405 nm with an ELISA reader (Filermax® F3, MolecularDevices). ELISA assay was performed for each bleed per animal inquadruplicate and repeated at least three times. Results are expressedas mean±standard deviations (SD). Coomassie stain and immunoblotanalysis of EBV glycoprotein gp350, gB, gp42 and gH/gL FC-His taggedrecombinant EBV glycoproteins were expressed in HEK-293 cells andpurified by protein A column. Results are shown in FIG. 9.

In vitro neutralization assay of EBV was performed to determineimmunized rabbits' sera neutralizing antibody responses. Day 49 pooledsera from n=6 from each treatment groups were serially diluted (1:40,1:80, 1:160 and 1:320) and incubated with purified EBV-eGFP virus,followed by incubation with either epithelial cells (HEK-293) or B cells(Raji) for 1 hour at 37° C. The virus/sera mixture was removed, cellswashed three times and incubated for 48 hours and GFP+ cellsrepresenting EBV-eGFP infection were enumerated by flow cytometry. Serafrom the THE group was used as the negative control and to normalizeEBV-eGFP percent infectivity. Neutralizing activity elicited by the EBVLPs vaccine was comparable to those elicited by the positive control,wild type EBV. The neutralization activity of sera from purifiedrecombinant EBV gp350 ectodomain were significantly lower than those ofwild type EBV and EB VLPs.

Dendritic cells maturation pulsed with EB VLPs and their ability tostimulate CD4+ and CD8+ T cells were assessed. As shown in FIG. 11A,dendritic cells (DCs) were generated from monocytes isolated from humanPBMC samples. Monocytes were maintained in culture with 100 ng/ml GM-CSFand 25 ng/ml IL-4 for 7 days to induce differentiation into DCs.Differentiated DCs were stained with anti-CD14 (FITC), anti-CD83 (PE),and anti-HLA-DR (APC) and analyzed by flow cytometry before beingco-cultured with autologous purified T-cells. As shown in FIGS. 11B and11C, DCs pulsed with EB VLPs were co-cultured with autologous T-cellslabeled with CFSE (TC to DC ratio of 60:1). On Day 7 T-cells werere-stimulated with either LPS (100 ng/ml), a pool of 25 EBV-specificpeptides (50 ng/ml per peptide), or DCs freshly pulsed with EB VLPs (2.5μl). Cultures were analyzed after 24 hours for CD4+(BUV395) and CD8expression (CFSE), and IFN-γ production (AF-700). Brefeldin A wasincluded in the cultures for the final 5 hours before analysis to blockcytokines release. EB VLPs stimulated CD8 T cell proliferation comparedto unstimulated cells or cells stimulated with LPS or EBV peptides (datanot shown).

Example 6: Immunogenicity of VLPs in Humanized NSG-BLT Mice

FIG. 12 shows immunization and bleeding schedules of humanized NSG-BLTmice. The method was describe in the previous publication by Fujiwara etal.²⁵ 10-12 week-old humanized mice (n=5/treatment) from University ofMassachusetts Medical School were immunized intraperitoneally threetimes at Days 0, 28, and 42 with 50 μg of purified EB VLPs suspended in0.5 ml TNE buffer adsorbed to 500 μg aluminum hydroxide (alum) and 50 μgmonophosphoryl lipid A from Salmonella enterica serotype minnesota Re595 (MPL). Purified 50 μg of UV-inactivated EBV, or 25 μg of purifiedEBV gp350 ectodomain adsorbed to alum and MPL served as positivecontrol, while 0.5 ml TNE adsorbed to alum and MPL served as negativecontrol. To assess short-, mid-, and long-term nAb responses, mice werebled seven days pre-immunization, then bled at Day 14, 35, and 49 afterprimary immunization and then humanely euthanized for terminal bleedingat Day 60. Total protein of EB VLPs and UV-inactivated virus werequantified using Micro BCA™ Protein Assay Kit (ThermoFisher) per themanufacturer's instructions. The immune responses in immunized huNSGmice and hu-NSG-BLT mice, including immunoglobulin IgM and IgG antibodyresponses, CD4+ and CD8+ T cell responses, are monitored to evaluate thetherapeutic effects of the VLPs.

Example 7: Co-Expression of EB VLP Subunits

Modified vaccinia Ankara (MVA) has high safety profile, includingimmunosuppressed individuals. As shown in FIG. 13A, the first step isPCR amplification of EBV gp350-gB-gp42-gL-gH from PCAGGS using primerswith engineered restriction site for cloning into transfer vector. Thesecond step is ligation of EBV gp350-gB-gp42-gL-gH PCR fragment intopGEM transfer vector containing an mH5 promoter, multiple cloning site(MCS) and transcription termination signal (TS). The third step isinsertion of Kanamycin resistance marker gene (KanR) and a I-SceIrestriction site flanked by 50 bp gB duplicate sequence. The fourth stepis PCR amplification of mH-EBV gp350-gB-gp42-gL-gH-TS using primers with50 bp MVA duplicate sequence overhangs. PCR product is inserted intoMVA-BAC via a first Red recombination utilizing the 50 bp MVA overhangssequence. The fifth step is the Kan selection marker removed from thenew MVA construct by introducing a double-strand break at the I-SceIsite and followed by a second Red recombination between the 50 bp gBsequence duplication.

FIG. 13B shows an MVA-BAC construct (not to scale) illustrating the fivegene insertion sites (1) Del2, (2) IGR3, (3) G1L/18R) and (4-5) Del3,going in different orientations. Table 1 below summarizes 4 differentMVA-EBV constructs, including the gene insertion sites on MVA-BACvector. EBV genes were inserted in the following order: construct 1:G1L/18R (Wild type EBV-gp350-gB-gp42-gL-gH) and Del 3(26aa-NP-EBNA1-LMP2); construct 2: G1L/18R (EBV-gp350-gB-gp42-gL-gHVLPs) and Del 3 (M-NP); construct 3: G1L/18R (EBV-gp350-gB-gp42-gL-gHVLPs), Del 3 (26aa-NP-EBNA1-LMP2) and IGR3 (M); and construct 4: G1L/18R(EBV-gp350-gB-gp42-gL-gH VLPs), Del 3 (26aa-NP-EBNA1-LMP2), IGR3 (M) andDel 2 (BMRF2/BDLF2).

TABLE 1 MVA-BAC EBV Constructs Insertion Site Construct 1 Construct 2Construct 3 Construct 4 Del2 BMRF2/BDLF2 IGR3 M M G1L/18R Wild type EBV-EBV-gp350- EBV-gp350- EBV-gp350-gB- gp350-gB- gB-gp42-gL- gB-gp42-gL-gp42-gL-gH gp42-gL-gH gH VLPs gH VLPs VLPs Del3 26aa-NP- M-NP 26aa-NP-26aa-NP- EBNA1-LMP2 EBNA1-LMP2 EBNA1-LMP2

FIG. 14 is a schematic representation of the cloning strategy togenerate MVA-EBV constructs. Wild type EBV gp350-gB-gp42-gL-gH orEBV-gp350-gB-gp42-gL-gH VLPs and M-NP are PCR amplified from respectivedonor vectors using PCR with 50 bp overhangs homologous to the 5′ and 3′end of G1L/18R and Del3 gene insertion sites, respectively. En Passantcloning technique is utilized to ligate the Wild type EBVgp350-gB-gp42-gL-gH or EBV-gp350-gB-gp42-gL-gH VLPs and M-NP PCRproducts into the MVA-BAC vector containing the pBeloc11 construct and aGFP gene which will be used as an indicator of transfection efficiency.The resulting clone, MVA-EBV can be transfected into Baby hamster kidney(BHK)-21 cell or chicken fibroblasts cells (CEF) to produce the virusfor further infection/immunization. The MVA-expressing wild type EBVgp350-gB-gp42-gL-gH-EBNA1-LMP2 or MVA-EB-VLP vaccine can be subjected tobiochemical characterization and the immune responses tested in variousanimal models to determine correlation of immune protection (e.g. wildtype mice, wild type New Zealand white rabbits, huNSG immunized ornon-human primate model) or any other appropriate animal model.

REFERENCES

The references, patents and published patent applications listed below,and all references cited in the specification above are herebyincorporated by reference in their entirety, as if fully set forthherein.

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1. A virus-like particle (VLP) comprising two or more EBV envelopeglycoproteins and one or more T cell antigens.
 2. The VLP of claim 1,wherein the two or more EBV envelope glycoproteins include gp350, gB,gp42, gH, gL, gM, gN, BMRF2, BDLF2, BDLF3, BILF1, BILF2, and BARF1. 3.The VLP of claim 1, wherein the one or more T cell antigens includeEBNA1, EBNA2, EBNA3a, EBNA3b, EBNA3c, EBNA-leader protein and LMP2. 4.The VLP of claim 1, comprising gp350, gB, gp42, gH, gL, EBNA1 and LMP2.5. The VLP of claim 1, further comprising one or more of NDV structuralproteins.
 6. The VLP of claim 5, wherein the one or more NDV structuralproteins include fusion (F), matrix (M), nucleocapsid (NP).
 7. Apharmaceutical composition comprising a therapeutically effective amountof the VLP of claim
 1. 8. A pharmaceutical composition comprising atherapeutically effective amount of a single VLP comprising two or moreEBV envelope glycoproteins and one or more T cell antigens.
 9. Thepharmaceutical composition of claim 8, wherein the two or more EBVenvelope glycoproteins include gp350, gB, gp42, gH, and gL.
 10. Thepharmaceutical composition of claim 8, wherein the one or more T cellantigens include EBNA1 and LMP2.
 11. The pharmaceutical composition ofclaim 8, wherein the single VLP comprises gp350, gB, gp42, gH, gL, EBNA1and LMP2.
 12. The pharmaceutical composition of claim 8, wherein thesingle VLP further comprises one or more of NDV structural proteins. 13.The pharmaceutical composition of claim 12, wherein the one or more NDVstructural proteins include fusion (F), matrix (M), nucleocapsid (NP).14. The pharmaceutical composition of claim 7, further comprising one ormore adjuvants, or one or more pharmaceutically acceptable carriers.15.-16. (canceled)
 17. A method of preventing or treating an EBVinfection or a condition associated with an EBV infection comprisingadministering to a subject in need thereof the pharmaceuticalcomposition of claim
 7. 18.-19. (canceled)
 20. An expression system forco-expressing two or more EBV envelope glycoproteins including a singlevector inserted with two or more nucleic acid sequences that encode twoor more EBV envelope glycoproteins, linked by one or more linkingsequences, such that the two or more EBV envelope glycoproteins can beco-expressed simultaneously, self-cleaved and/or self-processed toassemble into one or more glycoprotein complexes.
 21. The expressionsystem of claim 20, wherein the one or more linking sequences includeone or more 2A sequences encoding 2A peptides that mediate ribosomalskipping.
 22. The expression system of claim 20, wherein the singlevector is inserted with a single promoter before the two or more nucleicacid sequences such that the single promoter controls the expression ofthe two or more nucleic acid sequences.